April 25, 2011

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CHARGE QUESTIONS: FIFRA Scientific Advisory Panel Consultation on 

“Integrated Approaches to Testing and Assessment Strategies: Use of New 

Computational and Molecular Tools” May 24-26, 2011

A. Charge Questions About OPP’s Strategic Vision “Integrated Approaches to Testing and 

Assessment Strategy: Use of New Computational and Molecular Tools”

EPA’s Office of Pesticide Programs (OPP) is committed to improving and transforming our risk 

assessment approach to stay abreast of the state of the science and take advantage of 

emerging technologies. The goals are to increase the efficiency, effectiveness, and accuracy of 

the testing and assessment process by enhancing our abilities to target the effects of concern 

and to base decisions on the information most relevant to the assessment. The strategy to 

attain this goal is to enhance and advance our ability to use an integrated approach to testing 

and assessment (IATA). OPP views IATA as a logical way to transition from the current paradigm 

of broad in vivo testing and assessment to a system of targeted testing based on a mechanistic 

understanding of the toxicity of a chemical. To use mechanistic data across various levels of 

biological organization to support chemical assessments, there must be a meaningful

translation and linkage of this information to adverse outcomes that are relevant to human and 

environmental health. To accomplish this, we are adopting the concept of an adverse outcome 

pathway (AOP) as the conceptual framework to organize knowledge in order to identify

linkages at different levels of biological organization so that more efficient, hypothesis-driven 

approach to chemical testing and assessment can be developed and supported (Ankley et al.,

2010; Schultz 2011; SAP 2010). Establishing the linkages and dose-response relationships 

between the key events1and the adverse outcome informs an understanding of the magnitude 

of change needed to trigger the adverse consequences of a biologic perturbation as it relates to 

dose, timing and duration of the perturbation, as well as the susceptibility of the host. We 

believe that by establishing the causal pathway we will be able to more reliably screen for 

potential adverse effects by quantifying such linkages to estimate effects to whole organisms or 

populations using data collected from lower levels of biological organization.

Development of the toxicological understanding across a range of AOPs, from their causal 

molecular initiating event and subsequent key events to the adverse outcome will establish the 

 

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“Key events” are biological events along a pathway that are obligatory and quantifiable steps leading to 

an adverse outcome. Pertinent observations include, for example, specific biochemical events, cell 

proliferation, perturbations in hormones or other signaling systems, receptor–ligand interactions. To 

establish a series of key events along the causal path, EPA, the International Programme on Chemical 

Safety and the International Life Sciences Institute (ILSI) have established a harmonized weight-ofevidence approach using criteria based on those described by Bradford Hill, taking into account factors 

such as dose-response and temporal concordance, biological plausibility, coherence and consistency. 

(USEPA, 2005; Meek et al., 2003; Seed et al., 2005; Boobis et al., 2006; 2008)
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necessary knowledge to streamline the pesticide testing and assessment to more thoroughly 

integrate, in a tiered fashion, existing data, computer modeling, and in vitro models diagnostic 

of AOPs to target in vivo testing. Furthermore, once an AOP is established the information can 

be used to develop new quantitative predictive tools and qualitative read-across methods. 

Thus, AOPs enable a shift from a chemical by chemical approach to a category based approach. 

The current AOP framework employs a simplified depiction of a linear progression of events 

across levels of biological organization that translate a molecular perturbation to an adverse 

outcome (assuming the perturbation is sufficiently severe). However, it is well recognized that 

AOPs actually operate as networks within a systems biology context and that various AOPs can 

share key events and interact with one another in a variety of ways. Reductionist 

experimentation has generally been characterized by a more linear mode of thinking. In 

contrast, because omics and HTS (viewed as a large suite or battery of assays implemented in a 

high-throughput manner) can efficiently cover greater breadth of molecular initiating events 

and/or key events and associate them with a given chemical, they are, perhaps, better 

equipped for promoting a network-view. Ultimately, understanding of responses to stressors 

and effectively predicting toxicity, in a systems biology context that operates across multiple 

levels of biological organization, will require the kind of network-thinking that HTS and omics 

promote. Consequently, effective integration of data from hypothesis-driven, focused, testing, 

omics, and HTS represents an important foundation for an enhanced IATA. 

The purpose of this consultation is to seek guidance from the FIFRA Scientific Advisory Panel 

(SAP) about OPP’s vision, strategy, plans, and initial efforts to incorporate advances in 

molecular science and emerging in silico and in vitro technologies (e.g., structure-activity 

relationships (SARs), quantitative SARs [(Q)SARs], mechanistically-based in vitro assays, high 

throughput screening (HTS) assays) as components of an enhanced IATA. OPP is requesting the 

Panel’s input on whether plans to utilize emerging tools and methods represent a logical 

progression that will ultimately achieve our vision of increased efficiencies, effectiveness, and 

biological relevance in the assessment and management of risk associated with the use of

pesticides. The Agency is also seeking input on a number issues specifically related to the 

development and evaluation of predictive tools and libraries of AOPs.

1) Does the Panel believe that OPP’s strategic vision clearly articulates a sound scientific basis 

to making the risk assessment process more efficient and informative? To what extent have 

we described the critical components (i.e., knowledge bases and databases of existing 

information, predictive tools (e.g., (Q)SAR, in-vitro methods, HTS assays and AOPs) and the 

progression of events (i.e., research and the use of research) to support the evolution from 

the current testing and assessment paradigm to an improved IATA of targeted tiered testing 

based on an understanding of AOPs (i.e., the molecular and cellular events leading to 

adverse effects at the whole organism level)? Are there any key science issues that the 

Agency has not captured that are necessary elements to consider in an IATA approach?
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2)

a) Given that there could be hundreds of AOPs and that varying levels of AOP information 

are needed depending on the decision being made (i.e., the tier of the risk assessment), 

please discuss principles for scientific acceptance (i.e., tools for gauging the reliability 

and robustness) of AOPs and the use of AOPs to move away from chemical by chemical 

approaches. 

b) OPP plans to evaluate new tools and information using available criteria and methods 

such as the OECD principles for (Q)SAR expert systems and the EPA-IPCS (International 

Programme on Chemical Safety) mode of action framework. Please discuss these and 

other criteria the Agency can use to reliably integrate predictive tools earlier in the 

process.

c) How can the foundation of knowledge generated through historical and on-going 

reductionist (i.e., making use of information from lower levels of biological organization 

to understand adverse outcomes), hypothesis-driven, biological experimentation be 

effectively mined and integrated with the capabilities of omics (including 

toxicogenomics, transcriptomics, proteomics, and metabolomics) and high-throughput 

screening to speed the discovery and development of AOPs and their subsequent 

acceptance as a credible foundation for predictive risk assessment? 

3) OPP is at the beginning stages of contemplating metrics to demonstrate success in achieving 

our vision. What is the Panel’s initial thinking regarding the concepts, considerations, and 

factors OPP should take into account as we evaluate our progress? What methodologies are 

best suited to measure success in achieving our vision? For example, what methods would 

you recommend to quantify the development of AOPs and the qualitative and quantitative 

application of AOPs in risk assessment?
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B. Charge Questions for the Case Studies

Case Study 1: “Use of Toxicogenomic Technology to Inform the Risk Assessment A Case Study: 

Propiconazole”

In this case study we began with the identification of an adverse outcome. We incorporated 

omic and molecular technologies to develop a mode of action (MOA) for propiconazole based 

on apical, omic and molecular endpoints. The challenge for the future will be to use diagnostic 

assays of Key Events MOA in the opposite direction, i.e., as a tool for predicting specific 

toxicological responses. This will require an increased understanding of adverse outcome 

pathways (AOP)/MOA, and most importantly the quantitative relationships among the key 

events within the pathway. We envision that diagnostic in vitro assays for early key events will 

be sufficient to predict toxicological outcomes, or limit the need for extensive in vivo studies. 

Over time, the building of libraries of AOPs will make greater use of existing data and enable a 

shift from chemical-by-chemical approaches to the formation of toxicological categories based 

on key events along the pathway rather than intrinsic chemical activity. Thus, “read across” to 

fill data gaps would be based on MOA/AOP information permitting the application of data from 

a chemical(s) that has been well-characterized to similar untested chemicals. 

1) Please comment on the use of omic and related technologies employed to develop and 

link Key Events to create the MOA/AOP of propiconazole. Was the resultant MOA/AOP 

for propiconazole logical and scientifically sound?

2) Are there other approaches/technologies that could be used to develop and link Key 

Events to develop MOAs/AOPs for propiconazole and/or chemicals? 

3) Please comment on the general utility of AOPs/MOAs or Key events to read across 

chemicals to provide a more defensible weight of evidence support for the traditional 

risk assessment approaches that OPP will continue to use in the near- and mid-term? 

4) Please comment on the potential use of omic and related technologies to inform AOPs 

in non-mammalian taxa for use in ecological risk assessment?
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Case Study 2: Use of an Adverse Outcome Pathway (AOP) to Inform Risk Assessment -

Triclosan as a Case Study

The case study with triclosan presents an example of development of an adverse 

outcome pathway (AOP) as the conceptual framework to organize knowledge and identify 

linkages at different levels of biological organization to support a more efficient, hypothesisdriven approach to chemical testing and assessment (Ankley et al., 2010; Schultz 2011; SAP 

2010). Establishing the linkages and dose-response relationships between the key events and 

the adverse outcome informs an understanding of the magnitude of change needed to trigger 

the adverse consequences of a biologic perturbation as it relates to dose, timing and duration 

of the perturbation, as well as the susceptibility of the host. In this respect, the magnitude of 

the change in thyroid hormone level and susceptibility of the adult and developing mammalian 

organism to adverse consequences resulting from this perturbation are relevant areas of 

consideration for this case study. We believe that by quantifying such linkages we will be able 

to reliably estimate effects to whole organisms or populations using data collected from lower 

levels of biological organization. 

The purpose of this consultation is to seek guidance from the FIFRA Scientific Advisory 

Panel (SAP) about the Agency’s use and interpretation of the in silico, in vitro, and in vivo

technologies as components of the adverse outcome pathway for thyroid hormone 

perturbation. The Agency is requesting your input on the following questions with respect to 

the Triclosan Case Study:

1) Use of the adverse outcome pathway concept to characterize the impact of triclosan on 

the thyroid hormone system promotes a research and risk focus on toxicodynamic and 

kinetic differences in thyroid homeostasis between (1) the study species (e.g.., rats) and 

the population of interest (e.g., pregnant women/fetus and young children) and (2) 

potential species similarities and/or differences in the molecular initiating events. 

Please comment on scientific knowledge and types of data considered to be informative 

in selecting appropriate factors for interspecies differences. 

2) Please comment on the factors most responsible for the variability of toxicokinetics and 

toxicodynamics of compounds affecting thyroid hormone homeostasis among humans 

(particularly susceptible subpopulations such as pregnant women and children). 

3) Benchmark dose analysis involves selection of a benchmark response (BMR). The 

consequences of a perturbation of a key event depend on the magnitude, timing, and 

duration of the perturbation, which in turn depends on dose. Subtle perturbations of 

key events may be damped out and have little effect on downstream biology due to the 

operation of homeostatic processes. Please comment on the current understanding of 

the magnitude of perturbation of thyroid hormone levels in humans that may be 

required to lead to adverse outcomes.